PROJECT SUMMARY The long-term goal of this proposal is to develop, validate and market robust, cost-effective technologies for highly multiplexed imaging of cells in liquid and solid biopsies at sub-cellular resolution as a means to diagnose disease, identify patients likely to benefit from specific anti-cancer drugs, and investigate mechanisms of action of cytoxic, targeted and immuno-oncology drugs. This will be accomplished by optimizing a recently developed method, tissue-based Cyclic Immunofluorescence (t-CycIF) that enables 20-60 channel imaging of a wide range of tumor types and normal tissues. t- CycIF uses commercially available antibodies and wide-field fluorescence imaging to obtain high resolution images of (i) formalin-fixed, paraffin embedded (FFPE) samples as routinely used by pathologists for diagnosis of disease in routine practice and in clinical trials and (ii) liquid tissues prepared using the RareCyte AccuCyte blood separation system. In contrast to other methods for high-dimensional imaging of tissues, t-CycIF is purely optical and therefore has excellent resolution. It is sufficiently robust and inexpensive that it can readily be implemented in translational research labs and clinical pathology cores. This STTR proposal focuses on turning an academic t-CycIF protocol into an industrial-grade standard operating procedure (SOP) on RareCyte?s hardware and software platform. This will involve multiple rounds of method development and optimization jointly by RareCyte and HMS scientists, engineers and pathologists culminating in a milestone test on immune cells in solid tumors and human blood. The test is expected to provide information about how best to combine small molecule and immunotherapy in treatment of melanoma, a problem of outstanding interest in cancer biology and precision medicine. Aim 1 focuses on process optimization, transfer of the t-CycIF protocol to RareCyte and optimization of staining protocols, imaging procedures and data analysis algorithms; preliminary studies have established multiple ways in which methods used by RareCyte could substantially improve the HMS t-CycIF protocol. Aim 2 focuses on validation of antibodies for immune profiling of human solid tumors and blood, including optimization of fluorophore combinations; this work will pave the way for Aim 3 and for creation by Rarecyte or its commercial partners of antibody kits for specific applications. Aim 3 focuses on a critical milestone test in which we will immune profile FFPE samples from multiple patients with metastatic melanoma before and after treatment with dabrafenib and trametinib (targeted MEK-RAF inhibitors used in patients with BRAFV600E/K tumors). We will quantify changes in tumor, stromal and immune cells with therapy and disease progression. A second milestone study will demonstrate the use of CycIF on immune cells in liquid biopsies as a complement to multi-parameter flow cytometry. The stretch goal of the current proposal is to match the very high multiplicity of imaging cytometry/CyTOF (60-100 channels) on tissues but using a purely optical method readily deployable in a clinical setting,